Research On The Enzymatic Synthesis Of Cefprozil In Nonaqueous Phase

As a kind of representative antibiotics among the second-generation non-ester oral cephalosperin, cefprozil has been widely used in clinical, whose domestic and international market demand is enlarging year by year. There are some shortages such as complicated process, long production cycle, severe environmental pollution and high cost ect. Enzymatic synthesis of cephalosperin is being an effective path by which the mild reaction, less steps, and cleaner production is realized, but high cost of enzyme, unstable activity and unaccommodated reaction system are still the pressing problems to solve. Therefore, it is very necessary to explore and research the novel efficient cefprozil synthesis technology.This article is based on the above background, the decomposition of a role with cefprozil enzyme screening, based on a single preferred methoxy polyethylene glycol chemically modified enzymes and compare the free enzyme and enzymatic properties of the modified enzyme differences, in order to enhance the stability of biological enzymes; finally study the best preparation enzymatic synthesis of cefprozil non-aqueous phase reaction system modification, expansion and modification enzymes in the synthesis of cefprozil in the non-aqueous phase enzyme-catalyzed field The application of scientific significance and has a positive real economic, social significance. Mainly the following results:Based on the protease enzyme paint pretreatment, carries cefprozil aqueous phase decomposition experiments to study the different enzyme protein decomposition of cefprozil ability to apply comprehensive selection of cefprozil reverse synthetic enzyme species. The results show that neutral protease ion exchange chromatography results showed that after pretreatment cleaning, chromatographic curve presents two completely separate basic protein peaks, both with protease activity. Laccase fermentation broth by centrifugation, ultrafiltration, dialysis desalination, appeared two components are not completely separated laccase by activity electrophoresis, contain paint activity. The test protease, laccase cefprozil have decomposition, the decomposition rate of the difference lies in the different alkaline protease highest efficiency of49.25%, the lowest papain decomposition efficiency as40.25%. Its trans-Cefprozil enzymes break down more easily, its lowest value decomposition rate of80.5%, the highest value reached99.6%, the average decomposition rate of95.5%.Single-methoxy polyethylene glycol chemical modification of neutral protease, the presence of significant factors affecting the rate of modification by orthogonal experimental design to obtain efficient access to the enzyme protein modification parameters:pH value of7, modified temperature was45In, enzyme concentration was1.8g/L, the reaction time was5.5h, a neutral protease under conditions that the average rate of51.9%modified, better repeatability. After laccase modified under appropriate conditions, the modification was48.3%, below the neutral protease activity loss and more, only57.4%of the original activity.A comparative study of single-methoxy polyethylene glycol-modified and free of neutral protease enzyme enzymatic properties differ. The results show that the optimum temperature of the modified enzyme was not changed, and the optimal pH value, compared with the free enzyme, the modified neutral protease, as compared with the free enzyme, the optimum temperature is not changed, still50In; pH9.0modified optimal pH. At the same temperature, with the processing time increases, the relative activity of the modified enzyme and free enzyme have declined, but modifying enzymes decline slowly than the free enzyme. At different temperatures, the processing time the same condition after the modification of the thermal stability of neutral protease improved. Modifying enzyme substrate affinity Km values1.204mg·mL-1, compared to the free enzyme compared to neutral protease retouched affinity for the substrate increased. Modifying enzyme year free enzyme improved stability in various organic solvents are, modifications in the relative activity of the enzyme glycerol system reached92.3%.Using a modified method of the modified catalyst of the neutral protease, the enzyme found in dimethyl sulfoxide, n-hexane, glycerol and the like in non-aqueous organic systems can catalyze trans formula7-amino-3-E-propenyl-3-cephalosporin-4-carboxylic acid (trans-APRA) and D-Hydroxyphenylglycine (HPG) directed synthesis of trans cefprozil (trans-cefprozil). DMSO relative yield of34.8%glycerol was35.2%, DMF was21.3%. Therefore, considering the substrate solubility and protease activity, selected mixed non-aqueous systems of dimethyl sulfoxide and glycerol.APRA and HPG was synthesized cefprozil, and using HPLC to detect their relative yield. The effects of the enzymatic reaction temperature, reaction time, amount of catalyst, the ratio of substrate addition and solvent addition ratio influence factors on the relative yield of the synthesis of the enzyme in the reaction. Combined with single-factor experiments, response surface design experiments to determine the optimal conditions for the non-aqueous phase catalytic synthesis of cefprozil:The optimal reaction conditions modified non-aqueous phase neutral protease catalytic synthesis of cefprozil added as substrate ratio APRA:HPG=1:2, enzyme dosage HPG:Enzyme=1:1, the ratio of solvent to add DMSO:Glycero=2:1,temperature30℃, the reaction conditions in the relative yield was63.6%.On the enzymatic synthesis of trans cefprozil characterize the structure and activity of the drug, LC-MS/MS results showed consistent synthesized trans cefprozil compared with standard molecular weight; antibacterial tests showed this product to Staphylococcus aureus aureus and Bacillus subtilis have some antibacterial activity, but not as good as oral cephalosporin antibacterial effects of propylene.